Spectroelectrochemical techniques were used to evaluate the redox potential of various hemoglobins under various experimental conditions. We use Ru(NH3)63+as a redox mediator, which exchanges electrons with heme iron through an outer-sphere mechanism. Use of this mediator offers the advantage that it does not act as an allosteric effector. Studies of Hb stabilized in varied conformations confirm previous reports that Hb in the high oxygen affinity (R state) conformation is typically more easily oxidized than in the low oxygen affinity (T state) conformation. Accordingly, Nernst plots for Hb show evidence of cooperativity, with redox potentials that are sensitive to T state stabilization by anionic effectors. Alterations of the redox potential that are independent of the protein's conformational equilibrium between R and T states are exemplified by imidazole interactions with Hb. Imidazole binds preferentially to metHb. Changes in the redox potential induced by imidazole are very different from the changes associated with binding of heterotropic anionic effectors. Effectors such as inositol hexaphosphate stabilize the T state, significantly lower cooperativity of the oxidation process, and reduce the ease of oxidation. In contrast, as the imidazole concentration is increased, Hb is more readily oxidized, and cooperativity is maintained. These marked differences make it possible to use spectroelectrochemistry to differentiate between redox changes brought about by conformational shifts and changes brought about by other alterations in the active site. © 1994.
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